The overall goal of this proposal is to provide a molecular understanding of the structure/function relationships and enzymic mechanism of endothelial-type nitric oxide synthase (eNOS). The mechanistic hypothesis to be tested is based on a 3/2 coupling model between the reductase and the P450 oxidase mediated by CaM/Ca+2. We propose that CaM fixes a specific orientation between the FMN and heme centers without a drastic change in their physical distance. We also hypothesize that tetrahydrobiopterin (H4B), in addition to its structural role, is involved in the redox steps of oxygenase catalysis. We further suggest that the intimate spatial relationship of the L-arginine, H4B and heme sites leads to mutual regulation of ligand binding to each site. These specific interactions are proposed to be different in the three NOS isoforms. Such differences, together with the low similarity in the CaM target in each NOS isoform, determine the rate-limiting steps and the observed turnover numbers in the individual isoforms. To test these hypotheses we propose to: (i). Prepare eNOS and the two individual domains with a full complement of redox centers and use stoichiometric and potentiometric titrations to characterize the relative and absolute redox potential of each redox center; (ii).Evaluate interactions among L.arginine, H4B and heme binding sites by spectroscopic and kinetic methods with combinations of natural ligands and analogs; (iii). Characterize the electron transfer sequence and kinetics in eNOS and its oxygenase and reductase domains, and evaluate the proposed mechanism and the regulatory roles of CaM and H4B using rapid scan stopped-flow, rapid-freezing EPR and rapid-quenching/HPLC analyses to monitor individual redox centers. The planned studies will yield integrated knowledge about how eNOS (and other NOS isoforms) function, and provide structural information useful for the design of selective pharmacological inhibitors to controlling pathophysiological events associated with NO.